R-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid and l-aspartic acid salt, process for the preparation thereof and pharmaceutical composition comprising the same for antimicrobial

ABSTRACT

Disclosed herein are R-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid and L-aspartic acid salt, process for the preparation thereof and pharmaceutical composition comprising the same for antimicrobial. Because the R-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid and L-aspartic acid salt is more soluble and less toxic and has less side effects as an antimicrobial agent than hydrochloride and the other salts (D-aspartate and phosphate) conventionally used, the salt may be useful for oral and injectable administration.

TECHNICAL FIELD

The present disclosure relates toR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid and L-aspartic acid salt, process for the preparation thereof andpharmaceutical composition comprising the same for antimicrobial.

BACKGROUND ART

Quinolone carboxylic acid derivatives are synthetic antibacterial agentswhich are well known to be useful for the treatment of infectivediseases in human and animals due to their potent and broadantimicrobial activities. Currently, quinolone-based antimicrobialagents such as norfloxacin, ofloxacin, ciprofloxacin, etc. are veryusefully applied for the treatment of human diseases, and theirefficacies are acknowledged. However, these medicines have the problemthat even though they show excellent antimicrobial activities againstGram-negative bacteria, they still show ordinary or relatively lowantimicrobial activities against Gram-positive bacteria. Accordingly,there have been various studies for solving such problems of existingquinolone-based antimicrobial agents, and as a result, sparfloxacinhaving improved antimicrobial activities against Gram-positive bacteriahas been developed. However, this compound still shows weakantimicrobial activities against Streptococci, methicillin resistantStaphylococcus aureus (MRSA) and other currently increasingquinolone-resistant strains. The above-mentioned strains are well knownas pathogens of respiratory infections. Therefore, there are increasingneeds for the development of improved quinolone antimicrobial agentswhich exhibit excellent antimicrobial activities against these strains.

In quinolone-based antimicrobial agents,R-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid exhibit excellent antimicrobial activities against Gram-positivebacteria, Gram-negative bacteria, methicillin resistant bacteria, andexisting quinolone-resistant strains.

In general, it is well known to those skilled in the art that an activeingredient used in a pharmaceutical composition should be highly solublein water or aqueous solution having a broad range of pH. Accordingly,the development of salts having excellent solubility is needed in orderto increase the bioavailability of theR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid.

Thus, the present inventors have described various salts ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid in Korean Patent Laid-Open Publication No. 2001-0029698. Examplesof such acids are inorganic acids such as hydrochloric acid, phosphoricacid, sulfuric acid, etc., organic acids such as methane-sulfonic acid,p-toluenesulfonic acid, acetic acid, citric acid, maleic acid, succinicacid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelicacid, lactic acid, glycolic acid, gluconic acid, galaturonic acid,glutamic acid, etc., and alkali metal ions such as sodium ions,potassium ions, etc. However, hydrochloride is usually used as apharmaceutically acceptable salt ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid.

L-aspartic acid was approved by the US FDA as a food additive, and theacid has been safely and widely used. L-aspartic acid is a stable andcolorless liquid that is not hygroscopic and corrosive and has stabilityin preparation because it is not toxic. It is so easy to treat the acidthat L-aspartic acid may be easily used in mass production. In addition,the acid is known to contribute to hepatic detoxification, assist inmineral absorption, enhance DNA and RNA metabolism and improveimmunocompetence. However, when L-aspartic acid is administered alone,the solubility and internal absorption is so low that only minor effectsare known to be exerted when the acid is administered even in excess.Therefore, L-aspartic acid has not been conventionally used as apharmaceutically acceptable salt.

Thus, the present inventors have performed research to develop a salthaving excellent solubility to improve the bioavailability of theR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-l-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid, prepared L- aspartate ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid, confirmed that the L-aspartate has better solubility as well asbetter physical properties such as stability, etc. than hydrochlorideand D-aspartate, and aspartic acid is easily dissolved in the form ofsalt and internally absorbed to have a lower toxicity than the othersalts, and made the present invention.

DISCLOSURE OF INVENTION Technical Problem

One object of the present invention is to provide L-asparate ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid.

Another object of the present invention is to provide a preparationmethod of the L-aspartate ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid.

Still another object of the present invention is to provide anantimicrobial pharmaceutical composition containing the L-aspartate ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid as an active ingredient.

Solution to Problem

In order to achieve the objects, the present invention providesL-aspartate ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid, a preparation method thereof, and an antimicrobial pharmaceuticalcomposition containing the same as an active ingredient.

Advantageous Effects of Invention

Because theR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid L-aspartate of the present invention is more soluble thanhydrochloride, is less toxic than the other salts (D-aspartate,hydrochloride, and phosphate), and has reduced side effects as anantimicrobial agent, the salt may be useful for oral and injectableadministration.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides L-aspartate ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid represented by the following Chemical FIG. 1.

ChemistryFigure 1

R-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid L-aspartate of the present invention may be crystal or amorphous,and more preferably a crystal form.

The present invention also provides a preparation method of theR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid L-aspartate of Chemical FIG. 1. Specifically, a preparation methodaccording to the present invention, as represented by the followingReaction FIG. 1, includes reactingR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid with L-aspartic acid in an inert organic solvent.

L-aspartic acid used in a preparation method according to the presentinvention has been widely used as a main ingredient of aspartame, is astable and colorless liquid that is not hygroscopic and corrosive, andhas stability in preparation because it is not toxic. It is so easy totreat the acid that L-aspartic acid may be easily used in massproduction. In addition, because the acid is known to contribute tohepatic detoxification, assistance in mineral absorption, andenhancement of DNA and RNA metabolism and improve immunocompetence, itis expected that side effects accompanied by the use of antimicrobialagents may be reduced.

The inert organic solvent used in the preparation method of the presentinvention includes ethyl acetate, methanol, ethanol, isopropanol,acetone, acetonitrile, hexane, isopropyl ether, water, etc., and ethanolmay be the most preferable among them.

A preparation method of the present invention will be specificallydescribed as follows. First,R-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid is dissolved in an inert organic solvent. The inert organic solventmay be preferably used in an amount equivalent to a volume (ml) 10 to 20times based on a weight(g) ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid.

0.9 to 2.5 equivalent weight, and preferably 1.0 to 1.5 equivalentweight of L-aspartic acid may be added to 1 equivalent weight ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid and may be reacted at 30° C. to 70° C., and preferably 40° C. to60° C. for 10 min to 5 hours, and preferably 30 min to 2 hours toprepareR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid L-aspartate.

R-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid may be prepared at a high yield of 82-83% or more by thepreparation method.

The present invention also provides an antimicrobial pharmaceuticalcomposition containingR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid L-aspartate of the Chemical Formula 1 as an active ingredient.

Furthermore, the present invention provides a method for treatingbacterial disease, including administering to a patient in need thereofa therapeutically effective amount of L-aspartate ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid represented by the Chemical Formula 1.

The present invention also provides a use of L-aspartate ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid represented by the Chemical Formula 1 in the preparation of anantimicrobial formulation.

L-aspartate ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid of the present invention was shown to have better solubility thanR-7-(3-aminomethyl-4-methoxyimino-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid, hydrochloride and D-aspartate thereof and to be about 2 times moresoluble, in particular, in distilled water than the hydrochloride (SeeTable 1). There was also little change in content of the salt indistilled water, meaning that its chemical stability was excellent (SeeTable 2), and it was determined that the toxicity was relatively low dueto a high lethal dose 50 (See Table 3). Furthermore, the salt exhibitedpharmacokinetics equivalent to that of hydrochloride in an in vivopharmacokinetic experiment (See Table 4).

Therefore, the L-aspartate according to the present invention may beuseful as an antimicrobial agent.

A composition containing the L-aspartate according to the presentinvention may be used in the form of a general medicinal preparation.

That is, the L-aspartate according to the present invention may beadministered in various dosage forms, orally or parenterally whenadministered in an actual clinical setting. Pharmaceutical preparationsmay be prepared by including one or more pharmaceutically acceptablecarriers in addition to an active ingredient. The pharmaceuticallyacceptable carrier may be used by including saline solution, sterilewater, Ringer's solution, buffered saline solution, dextrose solution,maltodextrin solution, glycerol, ethanol, and a mixture of one or morethereof, and other conventional additives such as antioxidants, buffers,bacteriostatic agents, etc. may be added if necessary.

Solid preparations for oral administration include tablets, pills,powders, granules, capsules, etc. These solid preparations may beprepared by mixing a compound with at least one excipients, for example,starch, calcium carbonate, sucrose, lactose, gelatin, etc. In additionto simple excipients, lubricants such as magnesium stearate and talc maybe used.

In addition, liquid preparations for oral administration includesuspensions, solutions, emulsions and syrups, etc. In addition to watercommonly used as a simple diluent and liquid paraffin, variousexcipients, for example, wetting agents, sweetening agents, flavors,preservatives, etc. may be included. Preparations for parenteraladministration include sterilized aqueous solutions, non-aqueoussolvents, suspending agents, emulsions, freeze-drying agents,suppositories, etc. Propylene glycol, polyethylene glycol, vegetableoils such as olive oil, injectable esters such as ethyl oleate, etc. maybe used as non-aqueous solutions and suspending agents. Suppositoriesmay include witepsol, macrogol, tween 61, cacao butter, laurin butter,glycerinated gelatin, etc.

Furthermore, the pharmaceutical composition of the present invention maybe parenterally administered, and the parenteral administration may beeffected by hypodermatic, intravenous or intramuscular injection. Toprepare a parenteral formulation, a solution or suspension may beprepared by mixing the compound with a stabilizer or a buffer in water,and a unit dosage form such as an ampoule or a vial may be prepared.

An amount of the L-aspartate according to the present invention may bepreferably included in the ranges from 0.1% to 50% by weight based on atotal weight of the composition. However, the above ranges are not to belimited to this, but may depend on the conditions of the patient, kindsof diseases, and severities of diseases.

A preferable dose of the L-aspartate according to the present inventiondepends on the conditions and body weight of the patient, the severityof the disease, drug forms, administration routes, and duration, but maybe appropriately selected by those skilled in the art. However, 0.01mg/kg to 10 g/kg a day, and preferably 1 mg/kg to 1 g/kg a day may beadministered. The doses may be administered once or several times a day.

MODE FOR THE INVENTION

Hereinafter, the present invention will be described in more detail withreference to the following examples and experimental examples. However,the following examples and experimental examples are provided forillustrative purposes only, and the scope of the present inventionshould not be limited thereto in any manner.

Example 1

Preparation ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid L-aspartate

23 ml of methanol and 23 me of water were added to 7.8 g ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid, to which was added 2.57 g of L-aspartic acid, followed by stirringat 45° C. for 1 hour. After the solid of the reaction mixture wasfiltered, 78 ml of ethanol was added to the solid. The mixture wasstirred at 5° C. to 10° C. for 2 hours, followed by filtration anddrying to obtain 8.5 g of the target compound (yield: 82%).

¹H-NMR(D₂O, ppm): 1.03; (d, 2H, J=4.00 Hz), 1.27; (m, 2H), 1.47; (s,3H), 2.67; (dd, 1H, J=17.59 Hz, J=8.80 Hz), 2.77; (dd, 1H, J=22.03 Hz,J=3.64Hz), 3.34; (s, 2H), 3.63; (m, 1H), 3.86; (m, 1H), 3.96; (m, 5H),4.67; (s, 2H), 7.66; (d, 1H, J=12.43 Hz), 8.54; (s, 1H).

Melting point (m.p.): 164.2° C.

Comparative Example 1

Preparation ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid D-aspartate

30 ml of methanol and 30 ml of distilled water were added to 10 g ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid, to which was added 3.3 g of D-aspartic acid, followed by stirringat 45° C. for 1 hour. After the solid of the reaction mixture wasfiltered, 100 ml of ethanol was added to the solid. The mixture wasstirred at room temperature for 3 hours, and the solid produced wasfiltered and dried to obtain 8.67 g of the target compound (yield: 65%).

¹H-NMR(D₂O, ppm): 1.06; (2H, d, J=4.04 Hz), 1.32; (2H, d, J=6.96 Hz),1.51; (3H, s), 2.73; (1H, dd, J=17.59 Hz, J=8.80 Hz), 2.83; (1H, dd,J=22.03 Hz, J=3.64 Hz), 3.38; (1H, s), 3.65; (2H, m), 3.90; (1H, m),3.99; (4H, m), 4.10; (1H, m), 4.71; (2H, s), 7.68; (1H, d, J=12.27 Hz),8.57; (1H, s).

Melting point (m.p.): 163.3° C.

Comparative Example 2

Preparation ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid

According to the method described in Korean Patent Laid-Open PublicationNo. 2001-0029698,

(+)-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid was prepared.

Comparative Example 3

Preparation ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid hydrochloride

According to the method described in Korean Patent Laid-Open PublicationNo. 2001-0029698,

(+)-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid hydrochloride was prepared.

Comparative Example 4

Preparation ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid methanesulfonate

*40 ml of trifluoroacetic acid was cooled to 5° C. to 10° C., to whichwas added 20 g of7-[3-(t-butoxycarbonylamino-methyl)-4-methoxyimino-3-methyl-pyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid, followed by stirring for 1.5 hours. 140 ml ofisopropanol and 3.1 ml of methanesulfonic acid were added to thisreaction mixture and stirred at the same temperature for 2 hours. Thesolid produced from the reaction mixture was filtered and dried toobtain 10.2 g of the target compound (yield: 51%).

¹H-NMR(D₂O, ppm): 1.05; (2H, d, J=3.84 Hz), 1.32; (2H, d, J=7.16 Hz),1.51; (s, 3H), 2.80; (3H, s), 3.38; (2H, s), 3.67; (1H, m), 3.98; (4H,m), 4.10; (1H, m), 4.70; (2H, s), 7.65; (2H, d, J=12.27 Hz), 8.54; (1H,s).

Melting point (m.p.): 193.3° C.

Comparative Example 5

Preparation ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid formate

50 ml of purified water, 50 ml of ethanol, and 1.1 ml of formic acidwere added to 10 g ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid, followed by stirring at 55° C. to 60° C. for 1 hour. Subsequently,the reaction mixture was filtered, and then 40 ml of ethanol was addedto the filtrate. The mixture was cooled to room temperature and stirredat room temperature for 4 hours. The solid produced was filtered,followed by drying to obtain 6.4 g of the target compound (yield: 57%).

¹H-NMR(D₂O, ppm): 1.03; (2H, d, J=4.04 Hz), 1.31; (2H, m), 1.49; (3H,s), 3.37; (2H, s), 3.62; (1H, m), 3.95; (4H, m), 4.03; (1H, m), 4.66;(1H, s), 7.61 ;(2H, d, J=12.43 Hz), 8.45; (1H, s), 8.50; (1H, s).

Melting point (m.p.): 195.4° C.

Comparative Example 6

Preparation ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid phosphate

8.04 g ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid was added to 41 ml of phosphoric acid and 41 ml of distilled water,followed by stirring at 55° C. to 60° C. for 2 hours. Subsequently, thereaction mixture was filtered, and then the filtrate was cooled to roomtemperature. To the filtrate was added 33 ml of ethanol, and then thesolid produced after the mixture was stirred at 5° C. to 10° C. for 1hour was filtered and dried to obtain 8.54 g of the target compound(yield: 85%).

¹H-NMR(D₂O, ppm): 1.06; (2H, d, J=6.04 Hz), 1.32; (2H, d, J=6.96 Hz),1.51; (3H, s), 3.38; (2H, s), 3.66; (1H, m), 3.99; (4H, s), 4.10; (1H,d, J=12.27 Hz), 4.70; (2H, s), 7.65; (1H, d, J=12.23 Hz), 8.54; (1H, s).

Melting point (m.p.): 165.4° C.

Comparative Example 7

Preparation ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid p-toluenesulfonate

53 ml of ethanol and 53 ml of distilled water were added to 6 g ofp-toluenesulfonic acid, to which was added 10.51 g ofR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid, followed by stirring at 55° C. to 58° C. for 2 hours.Subsequently, the reaction mixture was filtered, and then the filtratewas cooled to 5° C. to 10° C. 52 ml of ethanol was, added to thefiltrate, followed by stirring for 1 hour. The solid produced wasfiltered and dried to obtain 8.24 g of the target compound (yield: 55%).

¹H-NMR(D₂O, ppm): 1.06; (2H, d, J=6.44 Hz), 1.31; (2H, d, J=6.96 Hz),1.50; (3H, s), 2.35; (3H, s), 3.37; (2H, s), 3.66; (1H, m), 3.99; (4H,s), 4.11; (1H, d, J=12.1 Hz), 4.70; (2H, s), 7.31; (2H, d, J=8.44 Hz),7.63; (2H, d, J=8.04 Hz), 7.68; (1H, d, J=12.2 Hz), 8.58; (1H, s).

Melting point (m.p.): 187.2° C.

<Experimental Example 1> Solubility test

Each solubility (μg/ml) of theR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid L-aspartate, D-aspartate, methanesulfonate, formate, phosphate,p-toluenesulfonate, and hydrochloride andR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid prepared in the Example and. Comparative Examples was measured atroom temperature.

The results are shown in Table 1.

TABLE 1 Salt used Solubility (mg/ml) L-aspartate (Example 1) 189.78D-aspartate (Comparative Example 1) 118.64 Free form (ComparativeExample 2) 2.79 Hydrochloride (Comparative Example 3) 93.30Methanesulfonate (Comparative Example 4) 99.08 Formate (ComparativeExample 5) 27.52 Phosphate (Comparative Example 6) 57.79p-toluenesulfonate (Comparative Example 7) 5.78

As shown in Table 1, the free form ofR-7-(aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid was rarely soluble in water due to the solubility of 2.79 mg/ml,while the L-aspartate according to the present invention was excellentin solubility, which was 189.78 mg/ml. In particular, the solubility ofthe. L-aspartate according to the present invention was higher than that(118.64 mg/ml of D-aspartate which is the optical isomer. TheL-aspartate exhibited about 2 times higher solubility than thehydrochloride (93.30 mg/ml) and much better solubility than the othersalts in Comparative Examples. Thus, the L-aspartate according to thepresent invention is excellent in solubility and may be useful as amedicine.

<Experimental Example 2> Stability Test

30 mg of each salt of theR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacids prepared in the Example and Comparative Examples was dissolved in100 ml of distilled water, and then was subjected to stability test atroom temperature and at 60° C. The results are shown in Table 2.

TABLE 2 Content (%) Room 60? temperature (after 3 Salt used Initial(after 3 weeks) weeks) L-aspartate (Example 1) 98.1 98.0 87.4D-aspartate (Comparative Example 1) 97.8 98.0 90.3 Free form(Comparative Example 2) 97.3 96.8 34.6 Hydrochloride (Comparative 97.998.0 90.0 Example 3) Methanesulfonate (Comparative 96.4 96.8 89.6Example 4) Formate (Comparative Example 5) 89.7 89.7 75.1 Phosphate(Comparative Example 6) 98.1 97.9 91.2 p-toluenesulfonate (Comparative98.6 98.1 87.3 Example 7)

As shown in Table 2, there was little change in content of theL-aspartate according to the present invention at room temperature,meaning that it was chemically stable.

<Experimental Example 3> Toxicity Test-Single Dose Toxicity in Mice

The following experiment was performed in order to observe the degree oftoxicity of the L-aspartate according to the present invention.

Male ICR mice were used as experimental animals. 5 male ICR mice weredivided into 6 dose groups, respectively and fasted except for water for24 hours.R-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid L-aspartate prepared in the Example 1 and the other salts preparedin Comparative Examples were intraperitoneally administered once (10ml/kg) at 500, 250, 125, and 0 mg/kg, respectively to observe the lethaldose 50 (LD50) for 14 days. The results are shown in Table 3.

TABLE 3 Lethal dose 50 Salt used (mg/kg) L-aspartate (Example 1) 209D-aspartate (Comparative Example 1) 176 Hydrochloride (ComparativeExample 3) 176 Phosphate (Comparative Example 6) 176

As shown in Table 3, it is determined that the L-aspartate of thepresent invention was less toxic than the other salts (D-aspartate,phosphate, and hydrochloride) due to a higher lethal dose 50 than thoseof the other salts.

<Experimental Example 4> In Vivo Genotoxicities

The following study performed to obtain in vivo genotoxicities,micronucleus test of L-aspartate and hydrochloric acid salt in male miceto the following procedure.

After 24 hrs after end of twice intraperitoneal treatment of aspartateor of single intraperitoneal treatment of cyclophosphamide(CPA), allanimal were sacrifice and the changes on the number of polychromaticerythrocyte with one or more nuclei (MNPCE) were evaluate among 2000PCEs with PCE/((PCE-i-normochromatic erythrocytes(NCE)) ratio among 500erythrocytes for detecting possibility of cytotoxicity. The highestdosage used in the present study was selected as 60 mg/kg in a volume of20 ml using distilled water as vehicle because quinolone antibioticshave been showed positive results in mouse micronucleus test, and 30, 15and 7.5 mg/kg were selected using common ratio 2 in the present study,respectively. in addition, intact control and positive control(cyclophosphoamide(CPA) 70 mg/kg)groups were added.

In these result, the MNPCE number of aspartate were not detectedsignificant change in 30, 15, 7.5 mg/kg treat groupas compared withintact control, respectively. But in the case of hydrochloric acid salt,the MNPCE number were singificantly increase in 7.5 mg/kg tested group.

<Experimental Example 5> In Vivo Pharmacokinetic Test

The L-aspartate and hydrochloride according to the present inventionwere orally or intravenously administered to SD rats, respectively andblood samples were collected at a predetermined time to compare in vivopharmacokinetics. Pharmacokinetic parameters are shown in Table 4.

Then, Cmax: maximum drug concentration, Tmax: maximum drug concentrationtime, T_(1/2): drug half-life, AUCO-t: area under the plasmaconcentration-time curve from time zero to t hours, and AUCO-inf: areaunder the plasma concentration-time curve from time zero to infinity.

TABLE 4 PK parameters of the hydrochloride and L-aspartate according tothe present invention in rats after p.o. and i.v. administrationPharmacokinetic Hydrochloride L-aspartate parameter i.v. (10 mg/kg) p.o.(100 mg/kg) i.v. (10 mg/kg) p.o. (100 mg/kg) C_(max) (μg/ml) 4.46 ± 0.967.62 ± 3.93 4.18 ± 0.29 9.08 ± 4.50 T_(max) (hr) 0 2.13 ± 1.97 0 2.13 ±0.63 T_(1/2) (hr) 1.552.05± — 2.05 ± 1.55 — AUC_(0-t) 8.72 ± 0.33 44.42± 21.38 7.52 ± 0.22 52.68 ± 28.73 (μg · hr/ml) AUC_(0-inf) 0.4610.72± —9.32 ± 0.28 — (μg · hr/ml) BA (%)a — 50.96 — 70.05 * estimated value byback extrapolation(C_(O)) aThis result is calculated by ratio ofAUC_(O-T)

The pharmacokinetic parameters of the hydrochloride and L-aspartateaccording to the present invention showed in Table 4. Although thestatistical significance was not observed, the mean oral bioavailability(BA, %) of the L-aspartate of the present invention was relativelyhigher than that of the hydrochloride in rat pharmacokinetic study. TheBA values of the hydrochloride and L-aspartate according to the presentinvention were 50.96% and 70.05%, respectively.

<Experimental Example 6> In Vivo Tissue Distribution Test

An in vivo tissue distribution test was performed in order to confirmthe degree of drug distribution of the L-aspartate and hydrochlorideaccording to the present invention in each organ. After 8-week-old ICRmice were purchased and adapted in the laboratory, each salt was orallyadministered to the mouse at a dose of 100 mg/kg. The mice were bled andsacrificed at a predetermined time and each organ was drawn to measurethe concentration in each organ. After the administration ofhydrochloride and L-aspartate, each organ concentration over time, thearea under the corresponding time-concentration curve, and the ratio ofpermeation into tissue were shown in Tables 5 and 6.

Then, P ratio is a ratio of permeation into muscle and calculated asintramuscular AUC/intraplasmic AUC.

TABLE 5 Hydrochloride Time Plasma Liver Kidney Brain Lung Spleen ThymusHeart Testicle Muscle (hr) (μg/ml) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g)(μg/g) (μg/g) (μg/g) (μg/g) 0.5 15.51 160.68 90.76 2.51 82.80 101.8124.55 59.29 4.79 39.79 1 14.24 124.93 87.84 3.09 79.50 111.78 57.9561.22 8.36 52.03 1.5 12.09 112.52 75.49 2.72 73.65 107.26 60.77 52.6612.66 50.40 2 12.04 109.11 69.92 3.18 63.66 101.77 61.30 57.59 16.9850.82 3 6.24 68.98 42.99 1.99 38.75 59.75 38.37 25.52 14.22 28.30 5 4.3255.73 32.76 1.60 23.42 42.08 25.68 17.31 12.92 18.42 8 2.29 27.03 18.040.73 11.41 21.51 12.82 8.86 7.77 8.26 AUC_(0-t) 53.55 564.25 352.9214.63 299.50 463.83 258.57 224.61 90.91 210.10 P ratio — 10.54 6.59 0.275.59 8.66 4.83 4.19 1.70 3.92

TABLE 6 L-aspartate Time Plasma Liver Kidney Brain Lung Spleen ThymusHeart Testicle Muscle (hr) (μg/ml) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g)(μg/g) (μg/g) (μg/g) (μg/g) 0.5 11.59 127.50 79.15 2.28 76.28 91.6326.52 47.22 4.94 38.02 1 12.24 114.15 79.20 2.55 70.10 98.14 48.11 57.978.82 39.58 1.5 10.74 117.77 77.42 2.34 71.52 84.89 48.85 49.96 10.2037.37 2 8.47 85.44 62.18 2.57 52.69 67.28 40.10 36.69 12.92 36.70 3 6.3070.82 48.76 2.02 38.74 57.56 39.78 27.72 14.84 28.83 5 4.62 57.35 34.031.52 24.52 43.72 25.94 01.33 11.52 18.02 8 1.87 26.56 20.17 0.33 11.6223.37 12.79 8.72 6.97 6.93 AUC_(0-t) 47.44 533.23 353.00 12.82 285.27418.47 235.49 203.05 83.15 183.68 P ratio — 11.24 7.44 0.27 6.01 8.824.96 4.28 1.75 3.87

As shown in Tables 5 and 6, when the L-aspartate and hydrochlorideaccording to the present invention was orally administered, there was nobig difference in concentration of the drug to be distributed in organtissues and AUC according to the form of salt. It was confirmed that thelevels of the L-aspartate according to the present invention permeatedwere highest in the order of liver, kidney, brain, spleen, lung, thymus,heart, muscle, and testicle.

<Preparation Example> Preparation of Pharmaceutical Formulations <1-1>Preparation of Powders

Compound of Chemical Formula 1 2 g

Lactose 1 g

The ingredients were mixed and filled into sealed packaging to providepowders.

<1-2> Preparation of a Tablet

Compound of Chemical Formula 1 100

Corn starch 100

Lactose 100

Magnesium stearate 2

The ingredients were mixed and tabletted according to a conventionaltablet preparation method to provide a tablet.

<1-3> Preparation of a Capsule

Compound of Chemical Formula 1 100

Corn starch 100

Lactose 100

Magnesium stearate 2

The ingredients were mixed and filled into a gelatin capsule accordingto a conventional capsule preparation method to provide a capsule.

<1-4> Preparation of Injections

Compound of Chemical Formula 1 10/

Diluted Hydrochloric acid BP to pH 3.5

Injectable Sodium chloride BP Up to 1

The compound of Chemical Formula 1 was dissolved in a proper volume ofinjectable sodium chloride BP, pH of a solution produced was adjusted topH 3.5 with diluted hydrochloric acid BP, and its volume was adjustedwith injectable sodium chloride BP. After being sufficiently mixed, thesolution was filled in a 5 type I ampoule made from transparent glass,which was then molten such that the solution was packaged under theupper grid of air. An injection was obtained by autoclaving the ampouleat 120° C. for 15 min or longer.

1-10. (canceled)
 11. A composition comprising an aspartate salt of aquinolone antimicrobial agent.
 12. The composition of claims 11, whereinthe quinolone antimicrobial agent comprises norfloxacin, ofloxacin,ciprofloxacin, sparfloxacin, or combinations thereof
 13. The compositionof claim 11, wherein the quinolone antimicrobial agent comprisesR-7-(3-aminomethyl-4-methoxyimino-3-methyl-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylicacid.
 14. The composition of claim 11, wherein the aspartate salt of aquinolone antimicrobial agent is L-aspartate of a quinoloneantimicrobial agent.
 15. The composition of claim 11, wherein theaspartate salt of a quinolone antimicrobial agent is D-aspartate of aquinolone antimicrobial agent.
 16. The composition of claim 11, whereinthe composition further comprises a pharmaceutically acceptable carrier.17. The composition of claim 11, wherein the composition is formulatedfor oral or injectable administration.
 18. The composition of claim 11,wherein the composition is a liquid form.
 19. The composition of claim11, wherein the composition is a solid form.
 20. The composition ofclaim 11, wherein the composition comprises 0.1 to 50% of the aspartatesalt of a quinolone antimicrobial agent by weight based on a totalweight of the composition.
 21. The composition of claim 11, wherein theaspartate salt of a quinolone antimicrobial agent has water solubilityof at least 100 mg/ml at room temperature.
 22. The composition of claim11, wherein the aspartate salt of a quinolone antimicrobial agent has atleast 70% bioavailability.
 23. The composition of claim 11, wherein theaspartate salt of a quinolone antimicrobial agent has a reducedcytotoxicity than a hydrochloric acid salt of the quinoloneantimicrobial agent.
 24. A method of preparing a composition of claim11, the method comprising reacting the quinolone antimicrobial agentwith aspartic acid in an inert organic solvent.
 25. The method of claim24, wherein the inert organic solvent is ethyl acetate, ethyl acetate,methanol, ethanol, isopropanol, acetone, acetonitrile, hexane, isopropylether, or water.
 26. A method of administering to a subject acomposition of claim
 11. 27. A method of treating a bacterial disease,the method comprising administering to a subject in need thereof atherapeutically effective amount of a composition of claim
 11. 28. Amethod of administering to a subject a composition comprising anaspartate salt of a quinolone antimicrobial agent.
 29. A method oftreating a bacterial disease, the method comprising administering to asubject in need thereof a therapeutically effective amount of acomposition comprising an aspartate salt of a quinolone antimicrobialagent.
 30. The method of claim 29, wherein the bacterial disease is adisease of Gram-negative bacteria, methicillin resistant bacteria, orquinolone-resistant bacteria.